Structures at the nanometer scale are sometimes referred to as nanoparticles. One example of nanoparticles is Fullerenes, which are the third allotropic form of carbon and form nanoparticles that may be an empty cage or the cage may contain cargo. The latter cage form is usually termed an endohedral Fullerene. Nanoscale endohedral Fullerenes can be used to stabilize reactive species inside the Fullerene cage, as in N@C60 or Sc2C2@C84. In addition, doped endohedral Fullerenes offer electronic and magnetic properties and might also be applied to electronics and information processing. Endohedral Fullerenes also have potential for biomedical applications such as targeted drug delivery, as well as other application areas.
In one application area, for example, the ability of endohedral Fullerenes to sequester one or more metal atoms, which may be toxic, inside the Fullerene cage has led to a research effort aimed at exploring their potential as contrast-enhancing agents for magnetic resonance imaging. Contrast agents enhance the quality of MRI images, aiding in the detection and diagnosis of injuries or abnormalities in the human body. The leading commercial MRI contrast agents are gadolinium (III) chelates such as gadolinium-diethylenetriaminepentaacetic acid, also known by its brand name Magnevist. Gadolinium III (Gd3+) works so well because of its unique electronic structure—it is the only ion with seven unpaired electrons. Once injected into the body, Gd3+ can magnetically “tickle” water protons present in tissues, accelerating their relaxation between radio-frequency pulses. Faster relaxation leads to higher signal intensity and therefore greater contrast in the MRI images. Encapsulating the gadolinium inside a Fullerene cage might prove safer, and such endohedrals potentially offer additional advantages. For example, the trimetallic-nitride-containing endohedral Fullerenes can accommodate three metal atoms inside each cage, potentially offering a more potent agent.
But before such endohedral Fullerenes can be tested and used in vivo, they must be made water-soluble. All endohedral Fullerenes exhibit extreme hydrophobicity, which must be overcome for many applications, especially for in vivo medical applications. One way to overcome this problem is to attach hydroxyl groups to the outer surface of the Fullerene cage. Compared with Magnevist, a commercially available contrast agent, prepared polyhydroxylated Gd@C82 can provide as much as 20 times better signal enhancement for water protons at much lower gadolinium concentration.
Although promising, there are problems with C82 endohedral Fullerenes. Most studies of metallofullerenes have centered on C82 isomers primarily because their solubility allows them to be more easily separated from empty Fullerenes and purified using high performance liquid chromatography (HPLC). But studies of polyhydroxylated Gd@C82 in rats revealed a significant, and potentially harmful uptake of the material by the reticular endothelial system, such as the lung, liver, and spleen. A similar uptake pattern of a polyhydroxylated derivative of Ho@C82 has been observed in these tissues, as well as in bone.
According to the Unites States Environmental Protection Agency (2003), which is funding research on nanoparticle toxicity, there is a serious lack of information about the human health and environmental implications of manufactured nanomaterials, e.g., nanoparticles, nanotubes, nanowires, Fullerene derivatives, and other nanoscale materials. Little is known about the fate, transport, and transformation of nanosized materials after they enter the environment. As the production of manufactured nanomaterials increases and as products containing manufactured nanomaterials are disposed of, these materials could have harmful effects as they move through the environment.
Forthcoming study results, including those funded by the EPA, may not be encouraging for pharmaceutical applications of Fullerenes like imaging contrast agents, because the metal-containing agent must be excreted without long-term retention in tissues. Unwanted organ and tissue retention also applies to targeted drug delivery systems using endohedral Fullerenes, which are about one nanometer in diameter. Organ and tissue retention issues also raise environmental concerns in general when in vitro endohedral Fullerenes or carbon nanotubes containing a metal or a toxic substance are free-floating in the air and are either inhaled into the lungs where they are absorbed into the body, and/or are absorbed into the body through contact with the skin.
Another drawback of C82 endohedral Fullerenes is that they are difficult to make in large quantities and with high purity, which is necessary for pharmaceutical applications and non-medical applications, like nanoscale electrical circuits. Generally, the success rate of creating endohedral Fullerenes is only about 1:10,000. This very poor success rates also leads to high costs. Endohedral Fullerenes cost as much as $1,000 per gram, in comparison to empty C60 cages, which cost only about $30 per gram.
Metallofullerenes in the C60 family, such as Gd@C60, have not been seriously considered for pharmaceutical applications because its members are generally insoluble and air-sensitive. On the plus side, M@C60 compounds can be produced in a carbon arc in yields up to 10 times higher than soluble M@C82 species. Water-soluble and fully air stable Fullerene fractions that are largely Gd@C60 have been experimentally produced. However, this material may contain several different isomers, rendering it unfit for many applications.
Besides using metal atoms, molecular clusters, and reactive species for medical imaging, chemists doing NMR spectroscopy have also encapsulated noble-gas atoms inside Fullerene cages and studied the interactions between the host and guest. In addition to Fullerene-caged helium, neon, argon, krypton, and xenon have also been put into Fullerenes, making unusual and highly stable noble-gas compounds in which no formal bond exists between the noble gas and the surrounding carbons. These compounds typically are made by heating the Fullerene in the presence of a suitable gas at 650° C. and 3,000 atmospheres. Under these conditions, though, no more than one in 1,000 Fullerene cages ends up with a noble-gas atom inside, making large scale production infeasible, as well as very costly.
Aside from helium for NMR spectroscopy, xenon (129Xe) is the only other noble-gas isotope having a spin of one-half, which makes the nucleus easily observable using NMR spectroscopy. But all endohedral Fullerenes suffer from a severe cargo carrying limitation, as the hollow core of the endohedral Fullerene is only seven to eight angstroms in diameter. Therefore, when trying to force xenon into C60, you get three to five times less xenon inside than helium, because xenon is so much larger. Such a tight fit brings into play another negative aspect common to all endohedral Fullerenes: the cage is highly conductive. When charge transfer to the Fullerene cage occurs, it distorts.
For example, xenon's 5p electrons are much closer to and interact much more strongly with the Fullerene's p electrons. When you alter the cage environment in any way, such as by making a Fullerene adduct, the cage may pucker slightly and the dimensions may change. The modified cage or the new group on the outside will interact very strongly with the enclosed cargo in ways that aren't easy to describe or predict. This cage distortion is not specific to xenon, and may occur with any enclosed particles that interact with the Fullerene cage. Endohedral Fullerene charge transfer and subsequent cage distortion is unacceptable in commercial and medical applications because results will not be consistent and predictable, and may also be harmful and injurious in some circumstances, like in vivo applications. This cage distortion drawback also potentially entails significant legal and medical liability issues.
The ability of endohedral Fullerenes to encapsulate various types of cargo is also limited. Apart from noble gases, the encapsulated metal atom can only be an alkali metal, alkaline earth metal, Sc, Y, U, or a lanthanide metal, with the most unusual of these species being Sc3N@C80, which has a nitride nitride-bridged Sc3N cluster inside a Fullerene. Most of the other metals in the periodic table do not form endohedral metallofullerenes, but rather form insoluble metal carbides and other unextractable materials.
Along with their limited cargo carrying capacity; charge transfer to the cage; organ and tissue retention; extreme hydrophobicity; and their difficulty of manufacture and very high cost, their cargo type limitations further limit the commercial and scientific potential of endohedral Fullerene-based endohedrals, for example, in the fabrication of nanoscale electronic integrated circuits.
Nanoscale integrated circuits from endohedral Fullerenes (“NICE”), apparently resolves at least one of these problems, namely, fabrication. NICE is the only known methodology that has been shown to produce macroscopic amounts of metal-containing endohedral Fullerenes. NICE uses a unique resistless proximal probe-based nanolithography technique to produce thin films of Fullerenes containing metal atoms. The films are characterized by laser desorption mass spectrometry and optical spectroscopy (IR and UV-vis absorption) among other methods. It is possible to dissolve the material and separate the endohedral compound from the empty Fullerenes and other material in the films. In this way macroscopic amounts of purified endohedral Fullerenes can be prepared, which up to now have been Li@C60. Material production and yield optimization for C60 endohedrally doped with other alkalis (Na, K) and the lanthanide La will also be developed at some point with NICE. A further innovative aspect of NICE is the additive approach taken to nanofabrication that uses a shadow mask technique whereby complex patterns such as rings and intersecting lines are readily produced. With the NICE method, the material composition of the as-deposited line can be varied, allowing for the formation of junctions within a single layer.
But NICE does not address the issues of limited cargo carrying capacity of endohedral Fullerenes, which is limited to just one to three atoms. Nor does NICE overcome the complex issues of charge transfer and endohedral cage distortion, which depend on certain quantum parameters of molecules, such as: point set groups, energies of electron levels, dipole (multipole) moments, electron affinity, ionization potential, molecular orbitals, electron density, electrostatic-potential derived charges, bond orders, net atomic charges, free valences, total energy, energy of formation, singlet and triplet UV/Visible spectra, IR and Raman spectra, polarizabilities, hyperpolarizabilities, magnetic moments, NMR properties, geometry optimization, atoms in molecules properties, etc. Nor does NICE overcome the issues of tissue and organ retention of potentially toxic endohedral Fullerenes when used in vivo, or the potentially harmful results of environmental exposure to endohedral Fullerenes. Nor does NICE overcome the extreme hydrophobicity of endohedral Fullerenes. Finally, NICE, which fabricates endohedral Fullerenes carrying metal cargo, does not fabricate Fullerenes that carry noble gases, and also does not overcome the fundamental cargo material type limitations of endohedral Fullerenes.
Methodologies such as NICE typically involve a “top down” assembly approach, and employ some form of lithography and replication. Top down approaches can be time consuming, expensive and exacting, and wasteful of materials if not performed correctly.
Another type of nanostructure, also sometimes referred to as a nanoparticle, consists of liposomes (spherical vesicles) that have been used as an alternative to in vivo endohedral Fullerenes because of the unique advantages of liposomes, which include their ability to protect their in vivo cargo from degradation, their ability to target their cargo, which can be a drug to the site of action, and to reduce the toxicity of side effects.
Another type of in vivo nanoparticle is comprised of lipids that has a surfactant agent and a cosurfactant agent and may also contain therapeutic agents, and possibly a steric acid. These lipid-based cages are 40 to 150 nanometers in size. These nanoparticles may be used to deliver entrapped agents across various biological barriers, such as the transmucosal passage, and also to overcome the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSG). As a general aspect, certain classes of surfactants have been shown to be effective at crossing these biological barriers and for allowing passage into the brain and CSF of various kinds of coated vesicles and nanoparticles having entrapped agents.
Another example of an in vivo nanoparticle is a therapeutic agent delivery system comprising a capsid formed from a coat protein of a bacteriophage selected from the group consisting of MS-2, R17, fr, GA, Q.beta, and SP, and with a foreign moiety enclosed in the capsid. The foreign moiety cargo is of a size sufficiently small to be enclosed in the capsid and the foreign moiety is linked to a RNA sequence comprising a translational operator of the bacteriophage. The translational operator binds to the coat protein during formation of the capsid.
The foregoing in vivo nanoparticle delivery approaches, including others in the prior art show promise as biological encapsulation methods and have the potential for becoming effective therapeutic agent delivery systems, especially those systems using surfactants. However, all of the foregoing biological cages, just like endohedral Fullerenes and others in the prior art, also suffer from various limitations that are unique to their various material compositions. For example, developmental work on liposomes and lipid nanoparticles has been limited, due to their inherent problems such as low encapsulation efficiency, rapid leakage of water-soluble drugs in the presence of blood components, and poor storage stability.
Furthermore, studies have concluded that the use of liposomes is an inefficient method of gene transfer for gene therapy, which incorporates functional genes into the cell to replace the action of dysfunctional genes. Inside of the body, liposomes fuse with cell membranes and deliver DNA to the cell via diffusion. However, studies have concluded that the use of liposomes is an inefficient method of gene transfer. Not only is plasmid size limited but gene loading is also poor: only one in every 100-10,000 liposomes contain intact DNA. In addition, many liposomes are taken into the cell by endocytosis, instead of releasing their DNA by diffusion. This leads to excessive breakdown of the DNA and results in poor transfection efficiency.
For many of these reasons, drug delivery systems using nanoparticles comprised of biodegradable polymers are emerging as one of the most widely used systems because of their numerous strengths, such as ease of fabrication, well-understood materials, and the ability to attach targeting moieties and barrier-passing surfactants.
Polymeric delivery systems were first used to provide the controlled release of many common drugs. Early polymers used were non-biodegradable and had to be surgically removed once its drug had been released. To avoid this inconvenience, researchers began searching for biodegradable alternatives. Successful controlled release systems have increased patient compliance in the administration of malaria drugs and several types of contraceptives in developing countries where patients have limited access to their physicians. In addition, such systems have also made improvements in the veterinary field, simplifying drug administration to animals. Controlled drug release has many advantages, including the ability to supply more constant drug levels, enable more efficient utilization of the drug, and the ability to locally deliver the agent and confine it to that area. In addition, decreased costs and frequency of administration add to the attractive features of biodegradable drug delivery systems.
Biodegradable polymers are generally divided into two categories: surface eroding polymers and bulk-eroding polymers. In surface-eroding polymers, erosion is confined to the polymer surface. In bulk-eroding polymers, erosion occurs throughout the entire cross-section of the polymer. However, the wide majority of polymers erode by a combination of both mechanisms. Degradation leads to erosion and is achieved by polymer chain scission, usually by hydrolysis.
For example, one biodegradable polymer nanoparticle approach uses sub-150 nm nanoparticles capable of transporting and releasing therapeutic agents, such as nucleic acids. DNA release in gene therapy applications is initially controlled by surface erosion followed by bulk erosion: as the backbone bonds hydrolize, channels form in the polymer allowing water to reach the interior of the nanoparticle. As water penetrates, bulk erosion occurs and the DNA is released. In one example, a biodegradable polymer nanosphere surface has attached to it a targeting moiety. In another nanoparticle embodiment, a biodegradable polymer nanosphere surface has attached to it a masking moiety. In yet another embodiment both targeting and masking moieties are attached to a nanosphere surface. In another biodegradable polymer example, surfactants have also been applied. For example, one nanoparticle mechanism uses biodegradable polybutylcyanoacrylate nanoparticles overcoated with polysorbate 80 for the purposes of crossing the BBB and CSF and delivering therapeutic agents.
The advantages and benefits of using biodegradable polymer nanospheres, including those that use surfactants and targeting moieties, are significant for in vivo targeted drug delivery. But they also create new classes of problems, and also do not overcome some existing ones, some of which issues are enumerated herein:
First, therapeutic agent models using in vivo biodegradable polymer nanospheres, liposomes, lipids, and caspid delivery nanoparticles, as well as endohedral Fullerenes, and others in the prior art still consist of an in vitro model being applied to an in vivo system, and clinical drug trials may show that promising in vitro results do not positively transfer to in vivo environment, especially in humans. This results in significant lost opportunity costs, as well as wastes large amounts of time, resources, and capital.
Second, side effect profiles are not satisfactorily addressed by the foregoing in vivo targeted delivery systems and in the prior art, and side effects may in fact be exacerbated because a highly potent concentration of a therapeutic agent will be delivered to highly targeted areas of interest. One possible consequence of in vivo targeted delivery systems is that dosing regimens, especially off-label use, may have to be significantly recalibrated by health care givers, necessitating new training and learning.
Third, the ability to cross various biological barriers into the brain and CSF, for example, using surfactants, and to deliver in vivo targeted concentrations of both small and large molecule payloads past these barriers will raise a host of new issues concerning agent efficacy, dosing and side effect profiles. As a consequence, individual patient factors such as genotype, phenotype, age, gender, ethnicity etc., may come into play more than ever, and these factors are not addressed by delivery systems in the prior art. Furthermore, once biological barriers to the brain and the CSF are commonly breached—especially by large molecule payloads that heretofore were not possible to typically deliver—new short and or long-term biological effects may also come into play and create important biological changes at the inter-cellular and intra-cellular level. Therefore, new, highly targeted drug regimens will need to be closely monitored and controlled after agent delivery for maximum efficacy and patient safety, and such monitoring and critical adjustments will need to be done on the fly and in vivo if they are to be maximally effective. However, all the foregoing in vivo delivery systems and others in the prior art lack such an in vivo ability to intelligently monitor, control, react, and dynamically adjust cellular processes after delivery of their agent payload to a target, as well as fail to take into account unique, individual patient factors.
Fourth, the materials comprising the foregoing delivery systems and others in the prior art are “dumb” materials. Although they may necessarily follow the control laws that regulate in vivo biochemical reactions and physiological processes, current in vivo delivery systems and others in the prior art do not feature or are not comprised of materials having the innate ability or characteristics to utilize and or leverage these control laws to intelligently respond to changing in vivo conditions. For example, the materials of the foregoing delivery systems and others in the prior art do not manifest an in vivo capability or the intelligence to dynamically alter a prescribed course of agent delivery in the face of an unexpected biological and or drug interaction, and in that sense, the regulatory control laws actually work against these dumb delivery systems. Once these dumb materials are set in motion, they cannot alter their course of behavior and are therefore highly static, fixed function systems.
Fifth, all the foregoing in vivo delivery systems and others in the prior art, with the possible exception of Fullerenes, lack structural persistence. Once the nanoparticles find their target and deliver their cargo, their job is finished and their various types of coatings rapidly disband, which means that the functionality of these various agent delivery systems and others in the prior art is severely time constrained. This temporal constraint represents a significant nanoparticle design limitation. Structural persistence for a period of time is a highly desirable quality for any nanoparticle or nanostructure as it permits the addition of temporal-based functionalities to the nanoparticles. For example, it may be highly advantageous for a nanoparticle to loiter for some period in an area after its initial agent delivery in order to monitor the situation and to potentially make a decision to deliver more agent cargo for improved agent efficacy.
Sixth, the ability to have multiple targeting moieties and one or more types of agents, while possible, is not currently practical with all of the foregoing systems, including others in the prior art. Multiple targets presume either on the fly smart target prioritization for a single cargo type or multiple cargo types that can be intelligently orchestrated and delivered in a dynamic environment—qualities that all of the foregoing delivery systems and others in the prior art currently lack.
Seventh, precise, highly ordered placement of cargo elements with minimal inter-cargo spacings is not possible with any of the foregoing in vivo agent delivery systems and others in the prior art, which are basically hollow nanospheres with no internal structural elements except for the cargo they may be carrying. Internal precision ordering of such agent cargo within the nanoparticle can, for example, enable the precise, intelligently controlled spatial and or temporal release of agents. Minimal inter-cargo spacings within the nanoparticle also afford the ability to tightly pack agents, especially mixed agents types—e.g., a diagnostic agent and a therapeutic agent—into the same nanoparticle with minimal interference between agents. Precision ordering and spacing within a nanoparticle is therefore in of itself an integral component of a targeted system, amplifying and extending the capabilities of agents carried within the nanoparticle.
Eighth, all current delivery systems and others in the prior art are limited to carrying cargo just within their cavities. Currently, they have no capabilities for building aggregated complexes of self-assembled structures that dynamically bind together one or more elements, some of which may be heterogeneous and external to the nanoparticle, into complex systems having one or more external elements, cavities and payload types. Current in vivo delivery systems and others in the prior art therefore do not make possible the assembly of sophisticated, complex nanostructures that fully exploit all the manifold possibilities of targeted agent delivery.
Ninth, there is no provision or capability for programming algorithmically-driven behaviors into the current targeted agent delivery systems and others in the prior art, with the possible exception of endohedral Fullerenes, and their capability for becoming smart, programmable and or self-directed systems to perform complex and sophisticated tasks in vivo is therefore severely limited, if not impossible.
Tenth, there is currently no provision or capability for integrating current agent delivery systems and others in the prior art, with the possible exception of endohedral Fullerenes, into other smart devices and mechanisms either in vivo or in vitro, either functionally or logically, including with other devices and operators at a distance (e.g., telemedicine), thereby limiting their overall therapeutic, diagnostic, therapeutic and system expansion capabilities.
Thus, there exists a need for an improved nano-structure element that overcomes the limitations of in vivo and in vitro endohedral Fullerenes, as well as overcomes the limitations of biodegradable polymer nanospheres, liposomes, lipid-formed systems, caspids and other agent delivery systems in the prior art for in vivo applications.